Difference between revisions of "Part:BBa K3866028"
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===Usage and Biology===
 
===Usage and Biology===
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This TU includes the <i>ygfH</i> gene placed under the control of the arabinosed-induced promoter <bbpart>BBa_K3866000</bbpart>. Initially, the level 1 cloning was performed using the trc promoter <bbpart>BBa_K3866001</bbpart>, though we weren't able to isolate a colony with the desired construct, as there is a possibility that the bacteria's metabolism went out of balance so no colonies survived to be chosen for screening. Instead we used the arabinose-indused promoter, which seemed to be working more than well.
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This TU includes the <i>ygfH</i> gene placed under the control of the arabinosed-induced promoter <bbpart>BBa_K3866000</bbpart>. Initially, the level 1 cloning was performed using the trc promoter <bbpart>BBa_K3866001</bbpart>, though we weren't able to isolate a colony with the desired construct, as there is a possibility that the bacteria's metabolism went out of balance so no colonies survived to be chosen for screening. Instead we used the arabinose-indused promoter, which seemed to be working as expected.
  
 
===Design Notes===
 
===Design Notes===
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===Verification of Cloning===
 
===Verification of Cloning===
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[[File:T--Thessaly--arac-ygfHgel.png|500px|thumb|none|<i><b>Figure 2.</b>: (C=Cut, U=Uncut) Restriction digestion of α1-sbm with: EcoRI, Expected bands: 6345bp, 2185bp, Positive result: C2</i>]]
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[[File:T--Thessaly--arac-ygfHgel.png|500px|thumb|none|<i><b>Figure 2.</b>: (C=Cut, U=Uncut) Restriction digestion of α2-ygfH (C5 - C6) with: EcoRV + HpaI, Expected bands: 4363bp, 1364bp, Positive result: C5</i>]]
  
 
===Experimental Use and Experience===
 
===Experimental Use and Experience===

Latest revision as of 10:56, 12 October 2021


ParaBAD:ygfH:Terminator

Usage and Biology

This TU includes the ygfH gene placed under the control of the arabinosed-induced promoter BBa_K3866000. Initially, the level 1 cloning was performed using the trc promoter BBa_K3866001, though we weren't able to isolate a colony with the desired construct, as there is a possibility that the bacteria's metabolism went out of balance so no colonies survived to be chosen for screening. Instead we used the arabinose-indused promoter, which seemed to be working as expected.

Design Notes

The coding sequence was domesticated. We removed BsmBI and BsaI sites in order to be compatible with GoldenBraid and MoClo. The sequence is cloned in seva α2 vector and has overhangs compatible for GoldenBraid cloning.

Figure 1. The level α module of the Propionate Production: α2:ParaBAD:RBS-ygfH-Double terminator

Verification of Cloning

Figure 2.: (C=Cut, U=Uncut) Restriction digestion of α2-ygfH (C5 - C6) with: EcoRV + HpaI, Expected bands: 4363bp, 1364bp, Positive result: C5

Experimental Use and Experience

This part showed functionality at the following parts: BBa_K3866030, BBa_K3866031

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2460
    Illegal BamHI site found at 1148
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 983
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 965


References

Akawi L, Srirangan K, Liu X, Moo-Young M, Perry Chou C. Engineering Escherichia coli for high-level production of propionate. J Ind Microbiol Biotechnol. 2015 Jul;42(7):1057-72. https://doi.org/10.1007/s10295-015-1627-4